We have divided this project into five portions. 1) Evaluation of SCL as a candidate oncogene. We have developed a transgenic mouse model of T-cell leukemia by crossing transgenic mice that overexpress SCL with transgenic mice that overexpress LMO1. Thymocytes obtained from clinically healthy SCL/LMO1 mice mice prior to the onset of a frank malignancy ("premalignant thymocytes"), show abnormalities in terms of thymocyte number, immunophenotype, proliferative index, and clonality. We recently discovered that over 70 percent of these SCL/LMO1 mouse tumors had mutations in the Notch1 gene, suggesting that Notch1 activation was a progression event that collaborated with SCL/LMO1 activation. The Notch1 mutations were not present in thymocytes harvested from 5 week old mice that had oligoclonal expansions of T-cells. The Notch1 mutations are likely to be important for maintaining the malignant phenotype of these cells, as T-cell lines established from SCL/LMO1 mice with Notch1 mutations are sensitive to gamma-secretase inhibitors. We have also used cre-lox technology to generate mice in which SCL can be activated conditionally. These mice show immunophenotype abnormalities similar to those seen in the SCL/LMO1 transgenic mice, with an increase in immature CD4-/CD8- cells and a decrease in CD4+/CD8+ cells. We have tumors from SCL/LMO1 mice for the presence of cancer stem cells. Surprisingly, preliminary results indicate that as few as 1 or 10 malignant cells from SCL/LMO1 mice can generate a tumor when injected into scid mice. 2) Several years ago, we cloned the BHLHB1 (OLIG2) and BHLHB2 (OLIG1) genes by virtue of their activation in patients with T-cell ALL. These genes encode bHLH proteins whose expression is normally limited to the central nervous system. We have shown that BHLHB1 is activated in a wide spectrum of malignant cell lines, and that greater than 50 percent of mice transgenic for both BHLHB1 and LMO1 develop T-cell tumors, demonstrating that dysregulation of BHLHB1 can be oncogenic. 3) We have cloned several chromosomal translocations that generate NUP98 fusion proteins. We have taken three complementary approaches to determine how these fusion proteins might be leukemogenic. In collaboration with Dr. Keith Humphries of the Terry Fox Laboratories, we have shown that mice reconstituted with bone marrow transduced with a NUP98-HOXD13 or NUP98-TOP1 retrovirus develop a myeloproliferative disease. Mice transduced with both NUP98-HOXD13 and MEIS1 develop AML. We have also generated transgenicmice that express NUP98HOXD13 or NUP98TOP1 in the hematopoietic compartment. The NUP98HOXD13 mice develop a highly penetrant myelodysplastic syndrome (MDS) that resembles the human disease in terms of peripheral blood cytopenias, dysplasia, apoptosis, and progression to frank leukemia. We have recently used retroviral tagging experiments to identify genes that collaborate with NUP98-HOXD13 in the process of leukemic transformation. We have also generated "knock-in" ES cells that express NUP98HOXD13 under control of endogenous NUP98 regulatory elements, but have been unable to generate chimeric mice from these ES cells. It seems likely that expression of the NUP98-HOXD13 fusion protein leads to a block in differentiation, as ES cells harboring the NUP98-HOXD13 knock-in allele are severely impaired in their ability to differentiate along hematopoietic lineages in vitro.4) Evaluation of CALM-AF10 as an oncogenic fusion. The CALM-AF10 fusion is caused by a t(10;11) chromosomal translocation seen in a wide spectrum of acute leukemia, but most commonly in T-cell ALL. We have generated transgenic mice that express the CALM-AF10 fusion in the hematopoietic compartment; some of these mice have recently developed T-cell ALL or AML.5) Zebrafish models.